1. Conformational Change in Proteins
Molecular Biophysics III
Prof. Daniel M. Zuckerman,
Dept. of Computational Biology

2. Conformational Change & Function
Many (most?) proteins function via conformational changes
Outline
Ensemble Picture and Examples
Hemogolobin and allostery
Myosin, kinesin and motion
Functional motif for NTP hydrolysis
Calmodulin and signalling
Reference/reading: Berg et al., Biochemistry
Also source for figures

3. Ensemble Picture (Statistical Mechanics)
An ensemble of paths, traversing ensembles of intermediate structures, connects two ensembles of structures
intermediates
structural ensemble A
structural
ensemble B

4. Motor proteins
Myosin

5. Fluctuations in Biology
Regulation: fatty acid binding proteins

6. Enzyme Conformational Change
Open/ligand-free
Adenylate kinase
Closed:
Ligand-bound

7. Conformational Change & Signaling
Signaling protein: Calmodulin
Calcium-free
Calcium-bound
Calmodulin, N-terminal lobe

8. More dramatic conformational flexibility
Open and closed Ca-bound calmodulin
Likely both occur in solution … and everything “in between”
Calcium-bound
Calcium-bound

9. Consequences of Induced Fit Idea
The idea: Ligand binding induces conformational change
Some possibilities:
Ligand binds to an apo-like or holo-like configuration
Ligand unbinds from holo-like or apo-like configuration
One way or another, proteins must undergo large conformational fluctuations
And this must happen all the time to allow constant binding and un-binding

10. Allostery: “cooperativity” in binding
For proteins with more than one binding site, the binding events often are not independent
Even when binding sites are identical!
Conformation & affinity change as additional ligands bind
This is allostery
Hemoglobin is the classic allosteric protein
Note: some of these states may not exist (stably).

11. Hemoglobin structure Four sub-unit homodimer (a,b)2 FYI:
Chien Ho at CMU

12. Hemoglobin: heme structure
Oxygen transported via integral heme groups
Four hemes, four binding sites
This small change triggers macroscopic motion

13. Binding-curve perspective
[oxygen]
bound oxygen
Fraction of

14. Physiological effects of cooperative oxygen-binding

15. Quantifying Allostery: Quasi-two-state model
MWC model (Monod, Wyman, Changeux)
Equilibrium between T, R -- each of which have four (static) identical binding sites
R = relaxed conf, T = tense conf., S = substrate
Conf. equil: R  T, with eq. const. L = [T]/[R]
Bind. equil. 1: R + S  RS1, with KR/4 = [R][S]/[RS1]
Bind. equil. 2: T + S  TS1, with KT/4 = [T][S]/[TS1]
Factor of 4 since 4 sites to bind
Further equilibria: TS1 + S  TS2

16. MWC Model can be “solved”
Solve with paper and pencil (no computer!)
Yields prediction for fraction of bound sites as a function of oxygen concentration
Highly successful for hemoglobin
Inadequate for some systems:
Omits sequence-dependence
Alternative model: KNF
Fersht, Ch. 10

17. Motor Proteins: Myosin
(kinesin)

18. Myosin structure
(ATP analog)
Binds to actin

19. Myosin: the structural trigger
Again, a tiny change triggers large-scale motion

20. Myosin-Actin Interactions
Figs from Alberts, Molecular Biology of the Cell

21. Kinesin structures
Kinesin expert at Pitt: Susan Gilbert (Biological Sciences)

22. Kinesin trigger

23. P-loop structural motif
For hydrolyzing NTP (to NDP)
N = nucleotide

24. Re-connect with statistical mechanics
Timescales and barriers
Rate as attempt frequency and Arrhenius factor
Multiplicity of pathways
Partial basis for ensemble picture (in addition to dynamic variability)
intermediates
structural ensemble A
structural
ensemble B

25. Structural Analysis of Calcium Signalling
Calmodulin is unusual
Ca2+-bound state is “open” -- solvent exposed
Hydrophobic residues exposed to solvent!
Contrast to enzymes which often “close” to envelop substrate
Calcium-free
Calcium-bound
Calmodulin, N-terminal lobe

26. Why CaM exposes hydrophobes
Hydrophobic surface bind other proteins to continue signalling cascade
Note: two conformational changes -- second is open-to-closed!

27. “Generalized Allostery”
Nussinov & coworkers in recent Proteins
Nearly all proteins can be considered allosteric
So long as interactions shift equilibrium
Calmodulin easily fits into this view
Calcium switches conformational equilibrium to open state
Open stat favors peptide binding